The invention relates to vaccines designed to protect against flaviviral disease. More specifically, the invention concerns recombinant envelope (E) glycoprotein produced in cellular production systems and formulated with modern adjuvants that are shown to maximize 1) induction of high titer virus neutralizing antibodies believed to be important in protection against infection and 2) provide protection of immunized animals from virulent viral challenge.
The family Flaviviridae includes the family prototype yellow fever virus (YF), the four serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4), Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE), and about 70 other disease causing viruses. Flaviviruses are small, enveloped viruses containing a single, positive-strand RNA genome. The envelope of flaviviruses is derived from the host cell membrane and is decorated with virally-encoded transmembrane envelope proteins. The E glycoprotein, which is the largest viral structural protein, contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. It is also a major target of the host immune system, inducing virus neutralizing antibodies and protective immunity, as well as antibodies which inhibit hemagglutination.
Although flavivirus transmission and the pathology of infection are quite varied among the different viruses, Dengue viruses serve as an illustrative example of the family. Dengue viruses are transmitted to man by mosquitoes of the genus Aedes, primarily A. aegypti and A. albopictus. The viruses cause an illness manifested by high fever, headache, aching muscles and joints, and rash Some cases, typically in children, result in a more severe form of infection, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), marked by severe hemorrhage, vascular permeability, or both, leading to shock Without diagnosis and prompt medical intervention, the sudden onset and rapid progression of DHF/DSS can be fatal.
Dengue viruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality with an estimated one hundred million cases of dengue fever occurring annually including 250,000 to 500,000 cases of DHF/DSS (Rigau Perez et al., 1998; Gubler, 1998). With the global increase in population, urbanization of the population especially throughout the tropics, and the lack of sustained mosquito control measures, the mosquito vectors of dengue have expanded their distribution throughout the tropics, subtropics, and some temperate areas, bringing the risk of dengue infection to over half the world""s population. Modern jet travel and human emigration have facilitated global distribution of dengue serotypes, such that multiple serotypes of dengue are now endemic in many regions. Accompanying this there has been an increase in the frequency of dengue epidemics and the incidence of DHF/DSS in the last 15 years. For example, in Southeast Asia, DHF/DSS is a leading cause of hospitalization and death among children (Hayes and Gubler, 1992).
The flaviviral genome is a single, positive-strand RNA molecule, approximately 10,500 nucleotides in length containing short 5xe2x80x2 and 3xe2x80x2 untranslated regions, a single long open reading frame, a 5xe2x80x2 cap, and a nonpolyadenylated 3xe2x80x2 terminus. The complete nucleotide sequence of numerous flaviviral genomes, including all four DEN serotypes and YF virus have been reported (Fu et al., 1992; Deubel et al, 1986; Hahn et al, 1988; Osatomi et al., 1990; Zhao et al, 1986; Mackow et al., 1987; Rice et al., 1985). The ten gene products encoded by the single open reading frame are translated as a polyprotein organized in the order, capsid (C), premembrane/membrane (prM/M), envelope (E), nonstructural protein (NS) 1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5 (Chambers, et al. 1990). Processing of the polyprotein is initiated cotranslationally, and full maturation requires both host and virally-encoded proteases. The sites of proteolytic cleavage in the YF virus have been determined by comparing the nucleotide sequence and the amino terminal sequences of the viral proteins. Subsequent to initial processing of the polyprotein, prM is converted to M during viral release (Wengler, and Wengler, 1989) and anchored C is processed during virus maturation (Nowak and Wengler, 1989).
While all dengue viruses are antigenically related, antigenic distinctions exist which define the four dengue serotypes. Infection of an individual with one serotype provides long-term immunity against reinfection with that serotype but fails to protect against infection with the other serotypes. In fact, immunity acquired by infection with one serotype may potentially enhance pathogenicity by other dengue serotypes. This is particularly troubling as secondary infections with heterologous serotypes have become increasingly prevalent as the virus has spread, resulting in the co-circulation of multiple serotypes in many geographical areas and increased numbers of cases of DHF/DSS (Rigau Perez et al., 1998; Gubler, 1998). Halstead (1982) demonstrated that anti-dengue antibodies can augment virus infectivity in vitro, and proposed that serotype crossreactive, non-neutralizing antibodies to E enhance infection in vivo, resulting in DHF/DSS (Halstead, 1981). This viewpoint is not however, universally accepted (Rosen, 1989). In particular the dengue gene product which may be responsible for the enhanced pathogenesis remains the subject of some debate. For example, it has been proposed that dengue serotype-crossreactive CD4+ CD8-cytotoxic T cells (CTLs) specific for NS3 may contribute to the pathogenesis of DHF/DSS by producing IFN-xcex3 and by lysing dengue virus-infected monocytes (Kurane et al., 1991; Okamoto et al., 1998; Mathew et al., 1998). Recent evidence demonstrating that CTLs specific for E are not serotype-crossreactive may suggest that use of E subunit vaccines would not induce the potentially harmful cross-reactive CTL response (Livingston et al., 1994). Other studies have suggested a potential role for NS1 in DHF/DSS (Falconer, 1997). Regardless of the mechanism for enhanced pathogenicity of a secondary, heterologous dengue infection, strategies employing a tetravalent vaccine should avoid such complications. Helpful reviews of the nature of the dengue disease, the history of attempts to develop suitable vaccines, structural features of flaviviruses in general, as well as the structural features of the envelope protein of flaviviruses are available (Halstead 1988; Brandt 1990; Chambers et al., 1990; Mandl et al., 1989; Henchal and Putnak, 1990; Putnak 1994; Rey et al., 1995; Rigau Perez et al., 1998; Gubler, 1998; Cardosa, 1998).
Although many approaches to dengue vaccines have been pursued, there is no acceptable vaccine currently available. While a significant amount of effort has been invested in developing candidate live-attenuated dengue vaccine strains, the strains tested to date have proven unsatisfactory (see, e.g., Eckels et al, 1984; Bancroft et al, 1984; McKee et al, 1987). Despite this limited success, live attenuated candidate vaccine strains continue to be developed and tested (Hoke et al, 1990; Bhamarapravati et al, 1987; Dharakul et al., 1994; Edelman et al., 1994; Angsubhakorn et al., 1994; Vaughn et al., 1996). The construction of several full-length infectious flavivirus clones (Rice et al., 1989; Lai et al., 1991; Sumiyoshi et al., 1992; Kapoor et al., 1995; Polo et al., 1997; Kinney et al., 1997; Gualano et al., 1998) has facilitated studies aimed at identifying the determinants of virulence in flaviviruses (Bray and Lai, 1991; Chen et al., 1995; Kawano et al., 1993; Cahour et al., 1995; Men et al., 1996; Hiramatsu et al., 1996; Pryor et al., 1998; Lai et al., 1998; Gualano et al., 1998; Valle and Falgout, 1998). While these studies remain quite preliminary and little information on virulence has been obtained, the cDNA clones derived from these studies are being used as a backbone for development of recombinant chimeric dengue vaccine strains (Bray and Lai, 1991; Chen et al., 1995; Bray et al., 1996; Lai et al., 1998). However, all of the live virus vaccine approaches remain plagued by difficulties in developing properly attenuated strains and in achieving balanced, tetravalent formulations.
Similarly, efforts to develop killed dengue vaccines have met with limited success. Primarily these studies have been limited by the inability to obtain adequate viral yields from cell culture systems. Virus yields from insect cells such as C6/36 cells are generally in the range of 104 to 105 pfu/ml, well below the levels necessary to generate a cost-effective killed vaccine. Yields from mammalian cells including LLC-MK2 and Vero cells are higher, but the peak yields, approximately 108 pfu/ml from a unique Vero cell line, are still lower than necessary to achieve a truly cost-effective vaccine product.
In the absence of effective live attenuated or killed dengue vaccines, a significant effort has been invested in the development of recombinant, dengue subunit or viral-vectored vaccines. Many of the vaccine efforts that use a recombinant DNA approach have focused on the E glycoprotein. This glycoprotein is a logical choice for a subunit vaccine as it is key to viral biology and the host immune response to the virus. The E glycoprotein is exposed on the surface of the virus, binds to the cell receptor, and mediates fusion (Chambers et al., 1990; Chen et al., 1996). It has also been shown to be the primary target for the neutralizing antibody response. Monoclonal antibodies directed against purified flaviviral E proteins are neutralizing in vitro and some have been shown to confer passive protection in vivo (Henchal et al., 1985; Heinz et al., 1983; Mathews et al., 1984; Kimuro-Kuroda and Yasui, 1988).
Although the primary amino acid sequence of flaviviral E glycoproteins are variable (45-80% identity), all have twelve conserved cysteine residues, forming six disulfide bridges, and nearly superimposable hydrophilicity profiles suggesting that they probably have similar secondary and tertiary structures. Recently, the structure of a soluble fragment of the tick-borne encephalitis (TBE) virus envelope glycoprotein was solved at 2 xc3x85 resolution (Rey et al., 1995). Coordinate synthesis of prM and E appears to be important to obtain the native conformation of E. Expression of E in the absence of prM may result in a recombinant product that presents a different set of epitopes than those of the native virion (Konishi and Mason 1993; Heinz et al, 1994; Matsuura et al, 1989). Epitope mapping of the E expressed with prM suggests that the co-expressed protein more closely resembles the native virus. As prM and E appear to form heterodimers during viral maturation and E undergoes an acid pH-induced conformational change, Heinz et al (1994) has suggested the association of prM and E is required to prevent irreversible pH-induced conformational changes during transit through the secretory pathway. However, it has been shown that carboxy-truncated forms of flavivirus E expressed in the absence of prM elicit protection from challenge (Men et al, 1991; Jan et al, 1993; Coller et al., unpublished data), suggesting expression of E in the absence of prM can result in the display of protective epitopes.
Recombinant dengue E glycoproteins have been expressed in several heterologous expression systems to date (See Putnak, 1994 and Chambers et al., 1997 for recent reviews). In general the systems have proven unsatisfactory for production of a cost-effective dengue vaccine due to limitations in antigen quality, quantity, or both. The following paragraphs highlight the major dengue recombinant subunit vaccine efforts and summarize the results obtained to date.
Most efforts using Escherichia coli have yielded poor immunogen incapable of eliciting protective responses. This probably reflects non-native conformation of dengue proteins expressed by bacteria and the necessity to process the viral proteins through the secretion pathway in order to achieve proper disulfide bond formation and glycosylation. While initial tests in mice suggested some level of efficacy (Srivastava et al., 1995; Mason et al., 1990), subsequent testing in primates failed to confirm the efficacy (R. Putnak, personal communication). Recently, fusion of a gene fragment encoding amino acids 298-400 (B domain) of the DEN-2 virus envelope was expressed as a fusion protein with the E. coli maltose binding protein (Simmons et al., 1998). This fusion protein conferred only partial protection to mice against challenge infection with a lethal dose of DEN-2 virus again demonstrating the limited efficacy of E. coli expressed products. Work in our laboratory has confirmed that recombinant products expressed in E. coli do not induce dengue specific immune responses in mice.
Use of the eukaryotic yeast expression systems Saccharomyces cerevisiae and Pichia pastoris for expression of dengue E has also proved to be less than desirable in terms of the quantities of immunogenic recombinant product obtained. Work in our laboratory has demonstrated that the expression levels of dengue E achieved in these systems are well below that which would be required to produce a cost-effective dengue vaccine. Work in other laboratories has suggested that the system may be useful for the production of virus-like particles (Sugrue et al., 1997). However, again the expression levels are quite low and the neutralizing antibody titer induced by the recombinant product is very low (1:10; Sugrue et al., 1997). This is despite the fact that the expression systems have been reported to be very efficient for heterologous expression of a variety of recombinant products (Cregg et al., 1993).
Use of the baculovirus expression system for flavivirus subunit vaccine production has also met with limited success (Reviewed in Putnak, 1994). In contrast to the high expression levels reported for various heterologous proteins in the baculovirus system, the levels of expression of flavivirus structural proteins were only moderate (Deubel et al., 1991; Staropoli et al., 1997). In addition, reactivity against a panel of anti-flaviviral monoclonal antibodies (MAbs) indicated that many conformationally sensitive epitopes were not present (Deubel et al., 1991). This suggests that folding of recombinant E produced in the baculovirus system may differ from the natural viral E protein. Furthermore, immunization with baculovirus-expressed recombinant envelope protein from DEN-1 (Putnak et al., 1991), DEN-4 (Eckels et al., 1994), DEN-2/DEN-3 hybrid (Bielefeldt-Ohmann et al., 1997), Japanese Encephalitis virus (McCown et al., 1990), or Yellow Fever virus (Despres et al., 1991) failed to elicit substantial titers of virus neutralizing antibodies or protection against viral challenge. Recently, Staropoli et al. (1997) were able to show neutralizing antibodies and some protection in mice using affinity-purified DEN-2 envelope as an immunogern. The truncated envelope glycoprotein was modified at the C-terminus with six-histidine amino acid residues (His6 tag) in place of the last 100 amino acids. However, this product induced only moderate neutralizing antibody titers and showed only partial protection in primates (Velzing et al., 1999).
Several reports have described vaccinia-flavivirus recombinants expressing envelope proteins as part of a polyprotein. The most consistently successful results in vaccinia expression of flaviviral proteins have been obtained co-expressing prM and E. Mice immunized with recombinant vaccinia expressing Japanese Encephalitis (JE) virus prM and AE developed higher neutralizing antibody titers and survived higher challenge doses of virus ( greater than 10,000 LD50; Konishi et al, 1992) than mice immunized with recombinant vaccinia virus expressing E alone ( greater than 10 LD50; Mason et al, 1991). Similarly, mice immunized with a vaccinia-Yellow Fever (YF) virus recombinant expressing prM-E were protected from virus challenge at levels equivalent to that of the attenuated YFV-17D vaccine, while vaccinia-YF virus recombinants expressing E-NS1, C-prM-E-NS1, or NS1 failed to protect mice (Pincus et al, 1992). Vaccinia-DEN-1 recombinants expressing prM-E elicited neutralizing and hemagglutination inhibiting antibodies in mice, while recombinants expressing DEN-1 C-prM-E-NS1-NS2a-NS2b elicited no E-specific immune response (Fonseca et al, 1994). The lack of recent advances in this technology have mainly been associated with the limited utility and problems associated with the use of vaccinia vectors for human immunization.
Use of the mammalian expression system based upon the Chinese hamster ovary (CHO) cell line to express dengue virus E has also been disappointing. Efforts in our laboratory and others has shown that although this is a very useful expression system for production of a variety of recombinant products (Bebbington and Hentschel, 1987; Cockett et al., 1990; Kaufman et al., 1985; Kaufman, 1990), DEN-2 E is not expressed efficiently. Intracellular expression levels of DEN-2 80%E were xe2x89xa6100 ng/ml and secretion was undetectable.
Thus, while many heterologous expression systems have been developed and shown to be effective for production of certain recombinant products, not all expression systems are effective for producing all recombinant products. In fact, despite the fact that a system may be reported to be effective for production of one recombinant protein, predictions on efficacy of expression of other recombinant products do not always hold. In particular, efficient expression of conformationally relevant recombinant flavivirus E has remained elusive. As described above a wide variety of expression systems ranging from bacterial, fungal, and insect to mammalian systems have failed to efficiently produce conformationally relevant flavivirus E in significant quantities, highlighting the highly empirical nature of efficient heterologous gene expression.
Within the last ten years an alternative eukaryotic expression system that uses the Drosophila melanogaster Schneider 2 (S2) cell line has been developed and used to efficiently express the envelope glycoprotein of Human Immunodeficiency Virus (Ivey-Hoyle et al., 1991; Culp et al, 1991; van der Straten et al, 1989). The inventions embodied herein make use of the Drosophila S2 system to produce recombinant flavivirus subunit polypeptides (specified by example are dengue and Japanese encephalitis E subunits), and have found the system can readily produce 20-50 mg of recombinant protein per liter of medium (unpublished). The recombinant product we have focused most of our efforts on is a soluble form of E, which is truncated at the carboxy-terminal end resulting in a polypeptide that represents approximately 80% of the full-length E molecule (amino acids 1-395; 80%E). We have expressed 80%E as a single open-reading frame with prM to enhance proper folding and secretion as described above. The expression levels achieved using this combination of expression system and recombinant DNA construct (20-50 mg/l) far exceed those achieved in other systems thus providing a cost-effective source of flavivirus antigen for vaccine production. In addition, we have demonstrated that the recombinant 80%E product secreted by these cells is conformationally relevant in that it binds to conformationally sensitive monoclonal antibodies and induces high titer virus neutralizing antibodies in mice and monkeys (Coller et al., in preparation).
Adjuvants are materials that increase the immune response to any given antigen. The addition of immune stimulating compounds to vaccines was first described in 1925 (Ramon, 1925). Since then a number of adjuvants have been developed, but currently only calcium and aluminum salts are licensed for use in human vaccine products. Although these adjuvants are sufficient for some vaccines, numerous studies have demonstrated that other adjuvants are significantly more efficacious for inducing both humoral and cellular immune responses and may be required to induce a protective response. Some of these, including Freund""s complete adjuvant (FCA) and Quil A, have been shown to be quite effective adjuvants in animals. However, they have significant toxicities or side-effects which make them unacceptable for human and veterinary vaccines. In fact, even aluminum hydroxide has recently been associated with the development of injection site granulomas in animals, raising safety concerns about its use. Because of these problems significant efforts have been invested in developing potent adjuvants which lack the undesirable side effects of adjuvants like FCA.
A number of modern adjuvant formulations have been developed and show significant promise, especially in combination with recombinant products. Several of these modern adjuvants are being tested in preclinical and clinical trials designed to examine both efficacy and safety. Several recent reviews summarize the approaches being used to develop modern adjuvants for human vaccines (Cox and Coulter, 1997; Gupta and Siber, 1995; Hughes and Babiuk, 1994). As described in these reviews there are five major modes of action of adjuvants; immunomodulation, presentation, induction of cytotoxic cellular responses, targeting, and depot generation. Various adjuvant formulations target different modes of action resulting in varying responses within the immunized animals. The specific formulation which is optimal for any given vaccine may be predicted based upon the disease target and desired response. However, often the optimal formulation must be determined empirically.
Adjuvants may be classified in a number of ways (Cox and Coulter, 1997; Gupta and Siber, 1995; Hughes and Babiuk, 1994). In broad terms there are two major categories, particulate and non-particulate. Included in the particulate category are aluminum salts, calcium salts, water-in-oil emulsions, oil-in-water emulsions, immune stimulating complexes (iscom) and iscom matrices, liposomes, nano- and microparticles, proteosomes, virosomes, stearyl tyrosine, and xcex3-Inulin. Non-particulate adjuvants include muramyl dipeptide (MDP) and derivatives (e.g. threonyl MDP, murametide, etc.), non-ionic block copolymers, saponins (e.g. Quil A and QS21), lipid A or its derivative 4xe2x80x2 monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), various cytokines including xcex3-interferon and interleukins 2 or 4, carbohydrate polymers, derivatized polysaccharides (e.g. diethylaminoethyl dextran), and bacterial toxins (e.g. cholera toxin or E. coli labile toxin). Often modern adjuvant formulations are combinations of various components designed to maximize specific immune responses.
The literature describing the use of various modern adjuvants in vaccine formulations is extensive. Many studies have demonstrated a potent immune response upon immunization with various recombinant products and modern adjuvant formulations using animal models (Reviewed in Vogel, 1998; O""Hagan 1998; Cox and Coulter, 1997; Gupta and Siber, 1995; Hughes and Babiuk, 1994). A few of the formulations appear superior in terms of efficacy and safety and are currently being tested in human clinical trials. However, the efficacy of any given adjuvant is immunogen dependent and thus predicting which combinations will be successful cannot be done reliably.
Among the most efficacious of the modern formulations are iscoms and iscom matrix (U.S. Pat. No. 5,679,354) which have demonstrated efficacy with influenza, respiratory syncytial virus, leishmaniasis, malaria, and HIV immunogens in animals (Andersson et al., 1999; Verschoor et al. 1999; Hu et al., 1998; Deliyannis et al., 1998; Papadopoulou et al., 1998; Bengtsson and Sjolander, 1996; Barr and Mitchell, 1996; Jones et al., 1995; Ronnberg et al., 1995; Takahashi et al., 1990). The Ribi adjuvant system, comprised of the detoxified endotoxin derivative, monophosphoryl lipid A, trehalose dicorynomycolate, with or without cell wall skeleton in a metabolizable oil, has also been demonstrated to induce potent immune responses in animal studies (Baldridge and Ward, 1997; Todd et al., 1997; Lipman et al., 1992; Johnson and Tomai, 1990; Kenney et al., 1989; Tomai and Johnson, 1989; Ribi et al., 1984). While these adjuvants have been shown to be effective with certain immunogens, there are also reports describing less than desirable effectiveness including non-protective responses with both iscom matrix (Echinococcus granulosus tegumental antigensxe2x80x94Carol and Nieto, 1998; herpes simplex virus glycoproteinxe2x80x94Simms et al., 1998; Leishmania major Parasite Surface Antigenxe2x80x94Sjolander et al., 1998) and Ribi adjuvant system (synthetic polypeptidexe2x80x94Deeb et al., 1992; Pasteurella multocida antigenxe2x80x94McClimon et al., 1994; trinitrophenol-hen egg albuminxe2x80x94Bennet et al., 1992; Smith et al., 1992). Thus, while the potential efficacy of a number of adjuvants are described in the literature, the optimal adjuvant for any given immunogen and any given disease target must be determined empirically.
We have demonstrated that the combination of certain modern adjuvants, in particular iscom matrix and the Ribi adjuvant system (RAS), with our Drosophila-expressed prM80%E flavivirus subunits results in an exceptionally potent vaccine formulation. iscom matrix is an immunomodulating agent that has an iscom-like structure and comprises within the iscom-like structure at least one lipid and at least one saponin, and a pharmaceutically acceptable vehicle, which sold under the trade name xe2x80x9cISCOMATRIX. This combination induces very high titer virus neutralizing antibodies in mice and monkeys and affords significant protection from viral challenge. However, combination of our recombinant prM80%E with other modern adjuvants failed to induce this potent immune response suggesting the uniqueness of the combination. Thus, the unique combination of flavivirus prM80%E expression constructs and the Drosophila expression system has allowed us to overcome the major limitations previously encountered in efforts to efficiently produce recombinant flavivirus subunit proteins. The addition of specific modern adjuvants to the recombinant product is effective in overcoming the final hurdle to production of an efficacious flaviviral recombinant subunit vaccine by significantly boosting the immune response in vaccinated animals. Examples illustrating the efficacy of the unique combination are contained herein below.
The invention provides vaccines containing, as an active ingredient, a Drosophila cell-secreted, recombinantly-produced form of truncated flavivirus envelope glycoproteins. The invention also includes a modern adjuvant as a critical component of the effective vaccine formulation The vaccines are capable of eliciting the production of neutralizing antibodies against flaviviruses and protecting against challenge with live virus. In the illustrations below, all products are expressed as a polyprotein including prM, and the mature recombinant 80%E products are secreted from Drosophila melanogaster Schneider 2 cells using the human tissue plasminogen activator secretion signal sequence (tPAL).
Thus, in one aspect, the invention is directed to a vaccine for protection of a subject against infection by a flavivirus. The vaccine contains, as active ingredient, the truncated envelope protein of a flavivirus and a modern adjuvant to modulate the immune response to the envelope protein. The truncated E is secreted as a recombinantly produced protein from insect cells. The vaccine may further contain additional truncated flavivirus E proteins similarly produced.